The Underlying Mechanism Of HBx Intervention On The Transactivation Of CYP3A4 In HBV-induced Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the fifth leading incidence and mortality cancer worldwide.Most of patients were dignosed with advacend stage of hepatocellular carcinoma,thus,5-year survival of these patients is less than 5%,which maily ascribes to lack of sensitive and specific biomarker for clinical early diagnosis of HCC.Chronic hepatitis involving in hepatitis B virus(HBV)infection is the major etiology factor for the initiation and development of HCC.In China,because of high HBV infection,the incidence of HCC induced by HBV is ranked first in the world.Hepatitis B virus x protein(HBx),a HBV-encoded transcriptional co-activator,plays an important role both in virus replication and in hepatocarcinogensis.As a multifunctional regulator,HBx could participate in multiple cell signalling pathways by protein-protein interaction to regulate transcriptional activation,cell cycle,DNA stability and apoptosis.Although previous literatures demonstrated that HBxAg could be detectable in patient serums,unfortunately,HBx still cannot be served as a biomarker for HCC diagnosis due to its short half-life.Therefore,it is necessary and significant to find out more stable and sensitive biomarkers in vivo for HCC early diagnosis,which could have consequently benefit on the prevention and chemotherapy of HCC.Drug-metabolizing enzymes such as cytochrome P450,which are responsible for the biotransformation and elimination of endogenous and xenobiotics,have come to be recognized as the first-line defense of liver from potential toxins and carcinogens.Among all of these enzymes,the first-pass of biotransformation was mainly carried out by CYP3A4,the most abundant isoform of monooxygenases,which governs the metabolism of over 50%therapeutic drugs and 30%endogenous substances.Compared to the parent compound,some metabolites of drugs will be more effectiveness,and some will be less toxicity after CYP3A4-mediate biotransformation.Overwhelming evidence demonstrated that dysfunction of CYP3A4 was closely related to the pathology of many liver diseases such as liver cancer,non-alcoholic fatty liver disease,hepatic fibrosis,cirrhosis,etc.Therefore,CYP3A4 could be served as a sensor for liver injure.However,either the expression level or the functional alteration of CYP3A4 in HCC patients,especially in HBV-induced HCC is not still clear.Three different aspects might address the mechanisms for the transcriptional alteration of CYP3A4.The first one is that epigenetic changes such as DNA methylation in 5-regulatory regions could directly result in transcriptional inhibition of CYP3A4 during the development of HCC,and same tendency are also abserved in histone methylation,acetylation and ubiquitination in CYP3A4 promoter.The secondary aspect is that HBx activate or suppress intercellular key pathways such as p53,?-catenin,CREB and NF-?B,which could indirectly mediate hepatocellular proliferation and the gene expression level of CYP3A4.Thirdly,it was reported that multiple transcription factors,especially the nuclear receptor(NR)family,play important roles in regulating the expression level of CYP3A4 by binding its promoter elements.PXR(Pregnane Xenobiotic receptor,NR1I2)and its heterodimerize partner RXRa(retinoid Xenobiotic receptor,NR2B1)were reported to be the predominant nuclear receptors responsible for transcriptional activation of CYP3A4.Recent studies revealed that HBx,co-located with PXR in nucleus,could directly interact with the ligand-binding domain of PXR and RXRa in HepG2 cells.Moreover,HBx could directly bind to the DBD domain of PPAR,which results in significantly suppression of PPAR-mediated gene transactivation by stereo-hindrance effect.However,other than activating signaling pathways,whether HBx could play a direct role in regulating the expression level of CYP3A4 through NR pathway is still unclear.Moreover,the relationship among HBx,PXR/RXRa and CYP3A4 is unclear for the development of HCC infected by HBV.Given high incidence of HBV induced HCC in China,and the predominant role of first-line defense mediated by CYP3A4,the present thesis would focus on demonstrating the underlying mechanism for the alterations of metabolizing activities,protein and mRNA of CYP3A4 in HBV-induced HCC,consequently,addressing the relationship of CYP3A4 with the development of HCC.1.The metabolism activity and expression of CYP3A4 were significantly decreased in HBV-induced HCCApproved by the Institute Research Ethics Committee and patient consents,ten HCC and the corresponding adjacent normal tissues were collected from Nanfang Hospital,the affinitied hospital of Southern Medical University.The diagnosis and stage classification wore conducted by utilizing pathological diagnostic techniques.The microsomes were prepared from fresh HCC and the adjacent normal tissues just after surgically resection.Kinetic parameters,indicating the enzymatic activities of CYP3A4,were determined by testosterone,a specific substrate of CYP3A4 by UPLC assay.Protein of CYP3A4 was determined by western blotting.Also mRNA of CYP3A4 was assayed by Real-Time PCR.All of specimens were classified as between Stage ? and Stage ?a by AJCC system.H&E staining showed no severe necrosis in HCC tumor tissues compared to the adjacent normal tissues.For determination of CYP3A4 metabolizing activity,the kinetics of testosterone metabolized into 6p-hydroxytestosterone was determined in commercial normal,the adjacent normal and HBV-induced HCC human liver microsomes(HLM),respectively.For Km and Vmax,no significant differences were observed between commercial normal(44.88 ?M,2512 pmol/mg protein/min)and the adjacent normal HLM(64.13?M,2162 pmol/mg protein/min),indicating the hepatocyte function of the adjacent normal HLM was similar to that of commercial normal HLM.However,the Km in tumor HLM(26.77 ?M)was significantly decreased when compared with that either in commercial normal or the adjacent normal HLMs.Moreover,the Vmax in tumor HLM(81.42 pmol/mg protein/min)was markedly lower compared to that of the adjacent normal HLM.The protein expression level of CYP3A4 was significantly decreased in most of HCC patients except P1 and P8.While the dramatically down-expression of CYP3A4 gene expression level(mRNA)was also observed in all HCC tissues.Overall,these above data suggested that the down-regulation of CYP3 A4 protein expression level,which was probably due to its transcriptional inhibition,would be the predominant causal factor attributing to the significantly decreased metabolic activity of CYP3A4 in HBV-induced HCC.2.The Protein and mRNA expressions of NF-?B(phospho-p65),p53(phospho-p53)and CREB(phospho-CREB)and GR,respectively,was inconsistent with CPY3A4 decrease;while only the protein expression level of HBx protein was negatively correlated with that of CYP3A4.Total RNA and total protein from HBV-induced HCC and the adjacent normal tissues were extracted the protein and mRNA levels of NF-?B,p53,p-CREB,GE,PXR and RXR?,were determined by western blot and Real-Time PCR.Significant individual differences were observed in the protein expression level of NF-?B(phospho-p65),p53(phospho-p53)and CREB(phospho-CREB)in the samples of HCC.However,no consistent alterations of each protein expression level were found in most of 10 samples.For example,compared to the adjacent normal tissues,the protein expression level of NF-?B(phosphate-p65)was only increased in the tumor tissues of P8 and P1,and that of p53(phospho-p53)only increased in P1,P3,P4 and P9.Also,higher protein level of CREB(phospho-CREB)was found in the nucleus of P3,P4,P6 and P9.While mRNA expression level of GR shows no significant differences in HBV-induced HCC tissues from PI,P2 and P4 patients compared to that of the adjacent normal tissues.Overall,the inconsistencies of NF-?B(phospho-p65),p53(phospho-p53),p-CREB(phospho-CREB),GR in protein and mRNA expressions of tumor tissues indicate these transcriptional factors may not be directly related with the decreased gene expression level of CYP3A4 in the HBV-induced HCC tissues.The basal protein expression levels of PXR and RXRa could modulate the transcription activation of CYP3A4 by protein-protein reactions.PXR/RXRa heterodimer could bind to the XREM region in ptomoter and enhancer of CYP3A4 gene to activate its transcription.We analyzed the nuclear protein levels of PXR and RXRa in HBV-induced HCC and the adjacent normal tissues.Compared with those of the adjacent normal tissues,no significant differences were observed in most of HCC samples except in P1,P3 and P8.At the meantime,the basal protein expression level of RXRa only elevated in P9.These results demonstrated that the protein level of each nuclear receptor could not be directly related with the down-regulated transcriptional activation of CYP3A4.On the other hand,mRNA levels of PXR were significantly decreased in most of tumor tissues compared to that in the adjacent normal tissues.3.HBx impairs the binding ability of PXR/RXRa in HBV-induced HCC.DNA-protein binding reactions were performed to determine the binding ability of PXR/RXRa in HBV-induced HCC and the adjacent normal tissues.The double-stranded 5'-biotinylated oligonucleotides of proximal-ER6,distal-DR3 and far-ER6 were synthesized as the PXR-RXRa heterodimer response elements of the CYP3A4.Chromatin immunoprecipitation assay(ChIP)and co-Immunoprecipitation assay were used to determine the intervention of HBx on the binding ability of PXR/RXRa heterodemier.Electrophoretic mobility shift assay(EMSA)was conducted to analyze the binding ability of PXR/RXRa heterodemier to three vital elements in the 5'regulatory region of CYP3A4:proximal-ER6(p-ER6,-365—+53),distal-DR3(d-DR3,-7.7kb—-7.2kb).The binding ability of PXR/RXRa was detected by the density and the shifting distance of shift bands,which showed the binding amount of heterodimers in the binding reaction and binding affinity to the promoter regions.Compared with that of the adjacent normal,the significant decreases of shifting distance in ER6(54.27%)and DR3(52.98%)showed that the PXR/RXRa complex formation and the binding affinity of PXR/RXRa to the promoter were dramatically impeded in HBV-induced HCC tissues.The supershift assay recognized by specific antibodies was used to unambiguously identify a potential protein presented in the protein-nucleic acid complex.The supershifted band of HBx and RXRa indicates that the direct role of HBx in the binding reaction of PXR/RXRa to the CYP3A4 promoter regions.Furthermore,statistically significant difference was observed in the correlation of the gene expression level of CYP3A4 and the binding ability,and the most correlation coefficient was found in proximal ER6 region.Therefore,EMSA results demonstrated HBx impairs the binding ability of PXR/RXRa to lead to the block of transcription of CYP3A4 gene in HBV-induced HCC.Interestingly,the intervention of HBx on the transcription of CYP3A4 gene was also confirmed by chromatin immunoprecipitation assay(ChIP)and Co-Immunoprecipitation assay(IP)because HBx could bind to PXR or RXRa when using HBx antibody for PXR or RXRa,respectively.Moreover,HBx had stronger binding to PXR or RXRa in the tumor tissues than those of normal tissues in HBV-induced HCC.4.HBx plays a predominate role in the down-regulation of CYP3A4 in HBx transfected hepatocarcinoma cell lines.By establishing the transient transfected cell lines,we further investigated the mechanism of HBx in disrupting the transcriptional activation of CYP3A4 in vitro hepatocarcinoma cell lines.When transfected with hPXR and hRXRa,the gene and protein expression levels of CYP3A4 were significantly increased in HepG2 and Huh-7,while a little down-regulation of CYP3A4 was also observed in HepG2.2.15,whereas the most reduction was only found with the presence of HBx when simultaneously transfected HepG2 and Huh-7 with hHBx.Otherwise,only the gene expression of CYP3A4,neither the PXR nor RXR?,was significant impede with the accelerated expression of HBx in nucleus,which implied that the HBx could involve in down-regulating the gene expression of CYP3A4 by not affecting the transcriptional activation of NRs.As expected,the metabolic activity of CYP3A4 was decreased by 97.53%and 59.52%,respectively,in HepG2 and Huh-7 transfected with hHBx;while down-regulation of 72.03%was observed in HepG2.2.15.In our further study,we analyzed the binding ability of PXR/RXR? to proximal-ER6 and distal-DR3,which was demonstrated that HBx significantly affected PXR response regions in previous data.Comparing with that of HepG2-hPXR/hRXR?,the binding ability of PXR/RXR? was decreased by 79.39±4.22%in HepG2-hPXR/hRXR?/hHBx;and 80.83%±2.43%down-regulation was also observed in Huh-7-hPXR/hRXR?/hHBx comparing with that of Huh-7-hPXR/hRXR?.Other than that,comparing 75%decreased binding ability in HepG2-hPXR/hRXR?/hHBx,only 50%reduction in HepG2.2.15-hPXR/hRXR?,indicating that HBx may exert stronger inhibition on the disheterodimerization of PXR/RXRa complex.The IP assay was conducted to validate whether HBx is the predominate protein responsible for the disheterodimerization in HBV-induced HCC tissues and transfected cell lines.The HBx antibody co-immunoprecipitated the PXR together with RXRa in HBV-induced HCC tissues,and the protein levels of PXR and RXRa in tumor tissues were positive correlation with that of HBx in nucleus.Meanwhile,recognized with RXRa,the prerequisite heterodimer partner for the transcriptional activation of CYP3A4,abundant protein level of HBx was also detected in tumor tissues comparing with that the adjacent normal tissue.To investigate whether the over-expression of HBx definitely present in heterodimer of PXR and RXRa,cells were transfected with hPXR,hRXR? and hHBx simultaneously.Comparing with the cells transfected with hPXR and hRXR? plasmids,large amount of PXR and RXR?,co-immunoprecipitated by HBx antibody was analyzed in HepG2-hPXR/hRXR?/hHBx,Huh-7-hPXR/hRXR?/hHBx,while less protein level of PXR was also observed in HepG2.2.15.Co-immunoprecipitated with RXR? antibody,the protein level of HBx was in proportion to the expression of HBx in cells.ChIP assay also showed that over-expressed HBx significantly inhibited the binding of PXR/RXR? to the promoter of CYP3A4 in HBV-induced HCC tissues,especially in p-ER6 region comparing with that in d-DR3.Therefore,the results transfected cell lines by HBx,PXR and RXR? cDNA demonstrated that HBx directly impairs the binding ability of PXR/RXR?,resulting in the block of transcription of CYP3A4 gene.5.HBx blocks the transcription of CYP3A4 as an oncogene in HBx transfected normal liver cell lines.By establishing the transient transfected normal liver cell lines,we further investigated the mechanism of HBx ib disrupting the transcriptional activation of CYP3A4 in vitro and the specific oncogenetic alterations of HBx.The protein and gene expression levels of CYP3A4 were significantly increased in L02 cell line when transfected with hPXR and hRXR? plasmids,which it gradiently impeded in CYP3A4 protein and mRNA with increasing concentration of HBx.When co-transfected L02-hPXR/hRXR? cells with 0.125?g hHBx,the gene and protein expression level of CYP3A4,respectively,was similar to that in L02-hPXR/hRXR?;while the protein level of CYP3A4 was significantly down-regulated even with the presence of 0.5 ?g hHBx,however,the most decreasing with 99.61±5.22%down-regulation were observed,respectively,on the gene and protein expression level of CYP3A4 in L02 transfected with lug hHBx.Compared with L02-hPXR/hRXR? transfected cells,significant decreasing of either band density or the mobility was found in L02-hPXR/hRXR?/hHBx transfected cells,even similar to that in HepG2-hPXR/hRXR?/hHBx.Furthermore,the HBx antibody recognized supershift band in L02 transfected cells showed the direct role of HBx in binding ability of PXR/RXR? in normal cells.When transfected L02 with increasing concentration of HBx,the binding ability was consistently decreased comparing with that of L02-hPXR/hRXR?,even 0.125 ?g hHBx may significantly altered the mobility.However,the band density was dramatically decreased only when transfected with 1 ?g hHBx,and the binding ability of PXR/RXR? was negatively correlation with the concentration of HBx in nucleus,while positively correlation with the gene expression level of CYP3A4.Other than that,we utilized the Co-IP and ChIP to further confirmed the predominate role of HBx in heterodimerization and binding ability alterations in transfected normal cell lines.Before the activation of other two critical transcriptional activate factors,either the gene expression level or the metabolic activity of CYP3A4 was able to significantly down-regulated in normal cell lines and even the morphology of L02 cells were significantly altered when transfected with hHBx,even similar to that of HepG2 and HepG2.2.15.In summary,data of the HBx in the transfected L02 cells showed that HBx has a dynamic interference on blocking the transcription of CYP3A4 gene,indicating the alteration of CYP3A4 in HBV-induced HCC development could reflect the effect of HBx on the contribution of HCC development.Taken together,the present study demonstrated the CYP3A4 metabolic activity was significantly decreased in HBV-induced HCC due to the marked decrease of the protein expression of the CYP3A4.The molecular mechanism may be that HBx,the key transcriptional co-activator for HCC development,directly impairs the transcription of CYP3A4 gene via blocking the binding ability and heterodimer formation of PXR/RXR? to CYP3A4 promoter.Interestingly,the interference of HBx on CYP3A4 transcription was in the mode of concentration-dependence.Thus,the transcription of CYP3A4 gene will be more severely blocked with the higher concentration of HBX in tumor cells.The potential mechanism implies that the alteration of CYP3A4 would express the progress and development of HCC induced by HBV.Therefore,CYP3A4 may be served as a senor or a biomarker for early diagnosis and prevention of HCC due to the HBV infection.At the same time,the data and results in the present study would be helpful and useful to the selection of therapeutic drugs for HCC.